Abstract B28: Downregulation of vimentin confers resistance to the microtubule stabilizing agents peloruside A and laulimalide in 1A9 human ovarian carcinoma cells

Abstract

Abstract Microtubule-targeting agents are an important class of drugs for the treatment of cancer. Although the development of drug resistance in cancer cells often limits the clinical efficacy of these agents, the mechanisms of resistance are not fully understood. Peloruside A (PLA) and laulimalide (LAU) are two marine sponge-derived stabilizing agents of microtubules. PLA and LAU represent the only known class of non-taxane site binding microtubule stabilizing agents and have a number of clinically useful potential advantages over paclitaxel, a widely used tubulin-targeting anticancer drug, including reduced susceptibility to the multidrug resistance P-glycoprotein efflux pump, good efficacy against taxane-resistant cells with mutations in the taxoid site, and more effective antitumor effects in non-small cell lung tumor xenografts. To understand the mechanisms of resistance to PLA and LAU, we used two drug-resistant sublines of the 1A9 human ovarian carcinoma cell line, R1 and L4. R1 cells are 6-fold resistant to PLA but remain fully sensitive to LAU; whereas, the L4 cells are 39-fold resistant to both PLA and LAU. We previously showed that both resistant cell lines exhibit different single point-mutations in their βI-tubulin gene. The L4 cells also have a high abundance of βII- and βIII-tubulin isotypes, which play a partial role in the resistance phenotype. To determine other protein changes that might contribute to the resistant phenotype of the R1 and L4 cells, we examined the proteome of the resistant cells using two-dimensional differential in-gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The proteomic analysis identified several proteins that were significantly altered relative to the parental 1A9 cells, including a decreased expression of vimentin in L4 cells. Using Western blotting and immunocytochemistry, the decrease in vimentin abundance seen in 2D-DIGE was confirmed. Vimentin levels were unchanged in the PLA-resistant R1 cells as well as two paclitaxel/epothilone-resistant sublines of 1A9 cells. Transfection with a full-length human vimentin cDNA partially restored sensitivity of L4 cells to PLA and LAU by 23% and 17% respectively. In vimentin-expressing parental 1A9 cells, tubulin-targeting and DNA-damaging agents caused degradation of vimentin and generation of vimentin degradation products. In conclusion, this study is the first to demonstrate a novel function for vimentin as a cellular determinant of the response to microtubule stabilizing agents, and that this effect is likely to be associated with the mode of cell killing by these agents. Since many of the proteins identified in this study have not previously been shown to play a role in drug resistance, our results also introduce potential new mechanisms by which cancer cells may become resistant to antimicrotubule agents.